The goal of this proposal is to produce comprehensive genomic resources for the fungal pathogen Histoplasma capsulatum. This resource will enhance and expedite the ability of H. capsulatum researchers to make key advances in the study of fungal pathogenesis. H. capsulatum, the etiologic agent of histoplasmosis, is a primary fungal pathogen that is endemic in the Ohio River Valley through the midwestern United States into Texas and is a leading pathogen affecting AIDS patients in the Midwest. This proposal represents a close collaboration between the Genome Sequencing Center at Washington University and biologists in the H. capsulatum community to create a fundamental resource for further exploration of H. capsulatum biology. Specifically, we will accomplish the following aims: 1. Construct a BAC clone-based physical map of the H. capsulatum genome, by fingerprinting each BAC and aligning overlapping BACs according to shared restriction fragment patterns. 2. Produce a highly accurate, complete genome sequence for Histoplasma capsulatum strain G217B with less than 1 error per 10000 nucleotides. The sequencing strategy used will entail a combination of paired-end plasmid and M13 sequencing reads from both whole genome shotgun and BAC-specific libraries. 3. Produce a reduced coverage (approximately 2-fold) genome sequence of the related Histoplasma capsulatum strain G186AR. The sequencing strategy used will entail paired-end reads from a whole genome shotgun library, with comparative assembly onto the existing G217B genome sequence. 4. Develop a microarray resource consisting of oligonucleotide sequences corresponding to each predicted gene in Histoplasma capsulatum strain G217B. Resulting arrays will be used to establish the relative expression level of each gene in both mycelial and yeast forms of the fungus. 5. Combine the resources developed in this project - BAC clone physical map, gene predictions and other sequence features, strain-specific DNA sequence variations, and microarray results - to fully annotate the resulting Histoplasma capsulatum genome sequence and encompass the information within an accessible database resource patterned after ACeDB.